Core Gene Expression and Association of Genotypes with Viral Load in Hepatitis C Virus (HCV) - Infected Patients in Punjab, Pakistan

Purpose: To determine genotypic distribution, ribonucleic acid (RNA) RNA viral load and express core gene from Hepatitis C Virus (HCV) infected patients in Punjab, Pakistan. Methods: A total of 1690 HCV RNA positive patients were included in the study. HCV genotyping was tested by type-specific genotyping assay, viral load, by real time polymerase chain reaction (PCR) and HCV core protein was expressed in E. coli. Antigenicity of core protein was confirmed by enzyme-linked immunosorbant assay (ELISA). Results: Out of total 1690 serum samples, type-specific PCR fragments were observed in 1482 (87.69 %) of the samples. In both genders, genotype 3a (55.44 %) was most prevalent followed by 3b (15.03 %), 1a (6.98 %) and 1b (3.14 %). Regionally, genotype 3a occurred most frequently in Jaranwala (59.72 %). Patients infected with genotype 3 had pre-treatment viral load values of 52.56, 15.79 and 31.65 %, while patients infected by other genotypes showed viral load values of 13.43, 35.27 and 51.3 % for low, intermediate and high categories of viral load, respectively. ELISA showed that core protein possessed greater antigenicity. Conclusion: HCV genotype 3a is the most prevalent genotype in Punjab, although the distribution of HCV genotypes in eight cities of Punjab was not uniform. HCV core protein used to develop local screening assays may be more effective than current commercial assays.


INTRODUCTION
The severity of HCV is well known all over the world.Prevalence of HCV is different in different geographical areas, which also depends on the different genotypes of HCV.These genotypes were developed from the high degree of HCV sequence variation.There are six major HCV genotype groups and seventy subgroups [1].In certain geographic areas Genotypes 1, 2 and 3 are more prevalent compared to 4, 5 and 6 which are present in other geographic areas.Genotypes 1 and 2 are predominantly present in the USA and Western Europe, genotype 4 found in Africa, genotype 5 in South Africa and genotype 3 in South East Asia, known genotypes do not have the same response to treatment strategies, so studying the geographic distribution of HCV genotypes is very helpful to plan treatment strategies [2].The other prognostic indicator is the pretreatment HCV viral load which is helpful in treatment decisions.High viral load was observed in some genotypes like genotype 1 as compared to other genotypes.Association of viral load and genotypes are not extensively studied but some association studies give the idea that high viral load is difficult to treat than low viral load [3].HCV virion is enveloped positive strand RNA virus.It consists of 10 viral proteins which are divided into structural and nonstructural proteins.Core is a structural multifunctional protein and it affects host cell functions, including apoptosis, HCV associatedsteatosis, immune cell functions, cell transformation, signal transduction, and transcriptional regulation leading to Hepatitis Cell Carcinoma [4].
Prevalence of HCV in Pakistan is 4.8 % which is among the in the world countries [5].Our study deals with the prevalence of HCV genotypes, viral load and expression of core gene in Punjab province of Pakistan.Punjab is the most populous province of Pakistan, with approximately 51.6% of the country's total population and 205344 total area in square kilometers [6].This is the most developed, populous, and prosperous province in Pakistan.
No study has been conducted in Punjab to evaluate the relationship between viral load and genotypes in HCV-infected patients.Since these two factors have significance in the treatment of patients, this study was conducted to determine genotypic distribution, RNA viral load and express core gene from HCV infected patients in Punjab, Pakistan .

EXPERIMENTAL Sample collection
Between the years 2007 and 2010, serum samples were collected from HCV-infected patients visiting collection centers/sub-centers of Citi Lab and Research Center, Lahore, Pakistan from eight different cities of Punjab, Pakistan.Data sheets with demographic characteristics, age, marital status and address from participating patients were filled by a research assistant.Patients were informed about the study and they gave their consent to participate in the study.The study was approved by the Ethics Committee of Citi Lab and Research Centre, Lahore, Pakistan (approval ref no.CLRC-143/11) and international guidelines for human studies were followed [7].

HCV RNA quantitation
RT-PCR of HCV RNA was performed using Mini OpticonTM System BIO-RAD Thermal Cycler.HCV RNA extraction and quantification Kits were manufactured by AJ Roboscreen Germany.The kit shows a linear measurement between 6,000 to 6,000,000,000 IU/ml and can detect < 172 copies/ml HCV RNA.Specimens yielding values more than upper limit were diluted 100 times, tested again, then results obtained were multiplied to this dilution factor to get the actual HCV RNA concentration in international units/ml.

HCV genotyping
HCV genotyping was performed with genotype specific primers based on PCR of Core region.cDNA was prepared with 50 ng of HCV RNA using 100 units of M-MuLV reverse transcriptase enzyme (Fermentas, Life Sciences, USA) at 37 °C for 50 minutes.For first round PCR amplification 2µl cDNA was used.As we had to detect different genotypes so type specific primers were divided into two groups on the basis of sizes of different bands so no genotype specific primers were of the same size in the same group on agarose gel.Each first round PCR specimen was used to perform two second round nested PCR amplifications.One with mix-1 primers and second with mix-2 primers in a reaction volume of 20µl.Mix-1 had genotype specific primers for 1b, 2a, 2b and 3b genotypes and Mix-2 had genotype specific primers for genotypes 1a, 3a, 4, 5a and 6a.Second round PCR products were observed on 2% agarose gel to separate type specific PCR fragment.DNA size marker 50bp DNA ladder (Fermentas, USA) was run on each gel.HCV genotype for each sample was determined by genotype specific PCR band.

Cloning of core gene HCV in expression vector pET21a (+)
pET21a (+) and core gene cDNA of HCV was digested thoroughly with Xbal and HindIII.The target gene and large fragment vector were purified by agarose gel electrophoresis.The 573bp target gene fragment was ligated with 5338bp linearized pET21a (+) plasmid vector by adding bacteriophage T4 DNA ligase overnight at 22 °C and stored at -20 °C.The ligated product was transformed into E. coli, and was incubated in an LB agar plate containing ampicillin overnight at 37 °C.Eight bacterial colonies were individually transferred into 2 ml of LB medium containing ampicillin in a loosely capped 15 ml tube, and the culture was incubated overnight at 37 °C with vigorous shaking.To confirm that the culture did contain the correct plasmid; we prepared a small amount of plasmid DNA and analyzed it by digestion with restriction enzymes and was also sequenced.

Identification of expressed proteins
Twelve percent sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) SDS-PAGE was used to identify the expressed proteins.The sample was prepared as follows: 1 ml of induced culture was centrifuged to collect the pellet, and the pellet was re-suspended and boiled for 5 min to lyse the bacteria with 2×SDS loading buffer.After electrophoresis, the polyacrylamide gel was stained with Coomassie Brilliant Blue, and scanned to analyze the expression level of the recombinant protein.The recombinant protein was used as antigen to test control positive sera by ELISA.

Statistical analysis
Data were analyzed using SPSS, version 16.
The results for all variables were given in the form of rates (%).Chi-Square or Fisher's exact test was applied to calculate p-value.P-values < 0.05 were considered significant.
Genotype distribution of HCV infected patients of different cities of the most populated province of Pakistan was evaluated (Table 2).Out of 617 HCV isolates of Lahore most frequent genotype was 3a in 351 (56.82 %) patients followed by 3b with 90 (14.59 %) patients.1a in Lahore was identified in 47 (7.62 %) patients followed by 1b in 17 (2.76 %).Only one isolate was identified of genotype 2a from Lahore.46 (7.46 %) patients

DISCUSSION
HCV is a major threat of the century; this is because of the nature of this virus which makes it difficult to treat.It leads to develop in severe conditions like liver cirrhosis and hepato-cellular carcinoma and also responsible in making people carrier of HCV.In India about 12-13 million HCV carriers were reported.With the change in the type of genotype, length of treatment and dosage of antiviral therapy can be changed.Viral response to interferon therapy is changed with genotype, such as genotype 3 and 1 gives better response.So HCV genotypes were very critically evaluated.The genotype of HCV varies along with geographical areas such as genotype 3a following genotype 1 was more prevalent in India in 2011 [8].
In the United States high prevalence of HCV subtypes 1a and 1b are well documented.In Europe HCV types 1b and 2, in Thailand,   [8].Some studies showed a close association of high viral load with advanced liver stage and some showed no relation with viraemia [14].
Here the core gene fragment of HCV genome was reversed transcribed and amplified successfully from the serum of patients from Punjab province of Pakistan.The fragment was sequenced and highly expressed in E.coli.
(Figure 5) The results of ELISA showed greater antigenicity of the recombinant protein, which demonstrated good prospects of using protein as an antigen to detect HCV antibodies.The nucleotide sequence homology analysis indicates the HCV isolates in this study (FR851292) belong to HCV core gene like Japan (D14309) and USA (EU099417).Previously this type of HCV was already isolated from this region [15].This gives the idea that for endemic HCV this ELISA based kit method is much better because of local recombinant protein [16].

CONCLUSION
The study indicates that genotype 3 is the predominant genotype in Punjab, Pakistan followed by genotype1.Baseline viral load is significantly high in patients with other genotypes (1a, 1b, 2a, 3b, mixed genotypes and undetermined genotypes) compared to genotype 3. Regional differences also exist for genotypes.Moreover, we have successfully expressed recombinant HCV core antigen.This antigen used to develop local screening assay may be more effective than current commercial assays.

Table 2 :
Distribution of genotypes of HCV isolates from different cities of Punjab, Pakistan (n = 1690) Note: UN = undetermined; percentages (%) in parenthesis; IS = insignificant

Table 4 :
HCV viral load categories and their distribution by gender and genotypes in different cities of Punjab, Pakistan